1. Field of the Invention
The present invention relates to a fluorescence-labeled (1→3)-β-D-glucan binding domain protein, a method for measuring (1→3)-β-D-glucan by fluorescence polarization using the fluorescence-labeled (1→3)-β-D-glucan binding domain protein, and an assay kit for carrying out the method.
2. Brief Description of the Background Art
A method for measuring degree of fluorescence polarization (Perrin, J. Phys. Rad., 1: 390-401 (1926)) has been utilized for measuring and analyzing trace amounts of biological substances and the like at high sensitivity using interaction among biological substances.
Examples of the interaction conventionally used include DNA hybridization, binding of a DNA binding protein with DNA, an antigen-antibody reaction, ligand-receptor binding, sugar-lectin binding, and binding of endotoxin and an endotoxin neutralizing protein (WO 98/21357).
On the other hand, a measuring method using a cascade reaction of a serine protease induced by the activation of a limulus blood cell extract (amoebocyte lysate) component by (1→3)-β-D-glucan which constitutes a fungal cell wall has been used for the detection of the presence or absence of mycotic infection which causes serious symptoms such as mycoses profundes and the like. However, limulus is an extremely valuable biological resource and the capture of limulus is regulated in certain regions.
It is known that a (1→3)-β-D-glucan sensitive factor (factor G) which binds to (1→3)-β-D-glucan, contained in limulus amoebocyte lysate, binds to (1→3)-β-D-glucan via a (1→3)-β-D-glucan binding domain in the α subunit, and its amino acid sequence and a DNA sequence encoding it have been revealed (WO 95/01432).
However, since changes in the degree of fluorescence polarization cannot be observed as a significant signal when there are no great changes in molecular weight and molecular structure by the binding reaction of a specific binding substance to an objective tested substance and a fluorescence-labeled the substance, it has been considered that the method is not suitable when the molecular weight of the specific binding substance be fluorescence-labeled is too large and the molecular weight of the test substance to be bound thereto is a degree of several thousand Da.
The limulus amoebocyte lysate as a valuable biological resource has a limitation in its amount for continuing its use for detecting fungi in the field of medical treatment and environmental hygiene which will continue to expand more and more in the future.